It is known that the production of useful foreign proteins in attempts to express large amounts of the foreign proteins depends on the copy number of genes, that is, the number of plasmids, and the strength of promoters which are used to control transcription. It was reported that the expression of target proteins can be increased by changing the number of plasmid vectors in individual bacteria, in addition to high-expression promoters which are conventionally frequently used (Tomio, M. et al., Appl. Microbiol. Biotechnol., 28: 170, 1988). Also, it was reported that RepE mutations in the mini-F plasmid can change the number of 25 plasmids in individual bacteria (Yasuo, K. et al., J. Biol. Chem., 267:11520, 1992).
Human papillomavirus (HPV) is estimated to infect more than 50% of adults worldwide. Particularly, four types of HPV including HPV 16, 18, 31 and 45 were reported to cause more than 80% of cervical cancer cases (Lowy, D. R. et al., Proc. Nat. Acad. Sci., 91: 2436, 1994).
Cervical cancer is the second most common cancer in women next to breast cancer, and according to WHO (World Health Organization), it is estimated that more than 500,000 new cervical cancer cases worldwide each year occur and more than 300,000 patients worldwide each year die from cervical cancer. Especially in developing countries, cervical cancer is a leading cause of death in women (Pisani, P. et al., Int. J. Cancer, 55: 891, 1993). The IARC report showed that the number of chronic HPV infection patients in developing countries is significantly larger than in advanced countries and that the most effective way to eradicate HPV infection is to administer HPV prophylactic vaccines.
The development of vaccines associated with cervical cancer has been focused on two types of prophylactic vaccines and therapeutic vaccines. The prophylactic vaccines aim to produce a stronger neutralizing antibody by HPV L1/L2 antigen, thus preventing a host from HPV infection, and preventing the disease from further progression, even if already infected. On the other hand, the therapeutic vaccine targets HPV E6/E7 and aims to induce specific cellular immune responses to degenerate formed lesions or malignant tumors.
Because the HPV E6/E7 protein is a cancer-specific antigen associated with the carcinogenesis of HPV-infected cells, studies on the use of the E6/E7 protein as a target for immune therapy of cervical cancer have been continued. Indeed, there is a report that when a HPV E6/E7 protein synthesized in a microbial system was administered to mice injected with tumor cells, tumor formation was inhibited or delayed (Gao, L. et al., J. Gen. Viol., 75: 157, 1994, Meneguzzi, G. et al., Virology, 181: 62, 1991). However, in the case in which live viral vaccines are used, problems associated with excessive viral replication can arise, similar to other cases. Thus, live virus vaccines are used only for research purposes in many cases and have disadvantages in that they take a long period of time to be commercialized and need considerable clinical trials.
Meanwhile, studies on the development of vaccines including bacterial vectors are also being actively conducted, and there is a report that HPV 16 VLP synthesized in attenuated Salmonella typhimurium induces the production of antigen-specific antibodies in the mucosa or whole body of mice (Denis, N. et al., Infection and immunity, 65: 3328, 1997). In the case of vaccines composed of synthetic peptides, only epitopes required to induce immune responses are synthesized for vaccination, and epitopes causing cytotoxic T lymphocyte (CTL) responses to HPV 16 E6/E7 have already been elucidated (Ressing, M. E. et al., J. Immunol., 154: 5934, 1995).
In addition to such attempts, studies on using transgenic vegetables themselves (obtained from vegetables including tomato and potato) as oral vaccines or edible vaccines to produce viral antigens in plants are being conducted. Typical examples thereof include hepatitis B surface antigen particles (Thavala, Y. F. and Artzen, C. J., Pro. Natl. Acad. Sci. USA., 92: 3358, 1995) and the capsid proteins L1 and L2 of papillomavirus (Korean Patent Registration No. 0366608). However, the plant systems have a problem in that the commercial use thereof is limited, because the amount of HPV L1 protein expressed is small and problems associated with purification occur.
Thus, in view of the fact that HPV-infected population is mainly concentrated on developing countries, the development of a method of preparing HPV antigens in a more economical and stable manner is urgently required in order to prevent and treat tumors derived from papillomavirus in the skin mucosa of oral cavities or genital organs.
The present inventors previously developed a vector for effectively expressing an HPV antigen protein on the surface of transformed recombinant microorganisms and a method for expressing an HPV antigen protein on the surface of the microorganisms (Korean Patent Registration No. 0609866).
Accordingly, the present inventors have made extensive efforts to develop a vector capable of more stably and constitutively expressing a high level of an HPV antigen protein on the surface of transformed recombinant lactic acid bacteria and, as a result, have found that the HPV antigen protein is more stably expressed at a high level in recombinant microorganisms transformed with a vector containing a repE mutant gene, thereby completing the present invention.